Method for the enzymatic production of 3-hydroxy-3-methylbutyric acid from acetone and acetyl-coa

ABSTRACT

Described is a method for the production of 3-hydroxy-3-methylbutyric acid by enzyme-catalyzed covalent bond formation between the carbon atom of the oxo group of acetone and the methyl group of a compound which provides an activated acetyl group. Also described are recombinant organisms which produce 3-hydroxy-3-methylbutyric acid, and related compositions and methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Phase of International Application PCT/EP2010/063460, which was published in English on Mar. 24, 2011, as WO 2011/032934, and claims the benefit of the filing date of European Patent Application No. 09170312.4, filed Sep. 15, 2009.

FIELD OF THE INVENTION

The present invention relates to a method for the production of 3-hydroxy-3-methylbutyric acid (also referred to as beta-hydroxyisovalerate or HIV) from acetone and a compound which provides an activated acetyl group comprising the enzymatic conversion of acetone and a compound which provides an activated acetyl group into 3-hydroxy-3-methylbutyric acid. The conversion makes use of an enzyme which is capable of catalyzing the formation of a covalent bond between the carbon atom of the oxo (i.e. the C═O) group of acetone and the methyl group of the compound which provides an activated acetyl group. Preferably, the enzyme employed in the process is an enzyme with the activity of a HMG CoA synthase (EC 2.3.3.10) and/or a PksG protein and/or an enzyme with the activity of a C—C bond cleavage/condensation lyase, such as HMG CoA lyase (EC 4.1.3.4). The present invention also relates to organisms able to produce 3-hydroxy-3-methylbutyric acid from acetone and a compound which provides an activated acetyl group and to the use of the above-mentioned enzymes and organisms for the production of 3-hydroxy-3-methylbutyric acid. Finally, the present invention relates to the use of acetone for the production of 3-hydroxy-3-methylbutyric acid.

BACKGROUND

3-hydroxy-3-methylbutyric acid (also referred to as beta-hydroxyisovalerate or HIV; see FIG. 1) is a metabolite of the essential amino acid leucine and is synthesized in the human body. It can be found in small quantities in grapefruit, alfalfa and catfish. It is also known to occur in some metabolic disorders of leucine catabolism, i.e. hypovaleric acidemia. It has been shown that 3-hydroxy-3-methylbutyric acid may have an effect on increasing muscle weight and strength (Nissen et al., J. Appl. Physiol. 81 (1996), 2095-2104). Wilson et al. (Nutrition & Metabolism 5 (2008)) proposes as the mechanisms of action the following:

-   -   increased sarcolemmal integrity via conversion by HMG CoA         reductase     -   enhanced protein synthesis via the mTOR pathway     -   depression of protein degradation through inhibition of the         ubiquitin pathway.

3-hydroxy-3-methylbutyric acid is supposed to help muscles combat protein breakdown, assist in muscle repair and support increased endurance. It has been described to help patients with chronic obstructive pulmonary disease in hospital intensive care units, muscle wasting associated with HIV and cancer and trauma victims with severe injuries. Thus, it is of commercial interest because of its use as a muscle enhancer for bodybuilding and as a medicament for avoiding muscle wasting.

U.S. Pat. No. 7,026,507 describes a process for preparing solid formulations of sodium 3-hydroxy-3-methylbutyrate in which, in a first process step, 4,4-dimethyloxetan-2-one is reacted with aqueous sodium hydroxide to form a solution of sodium 3-hydroxy-3-methylbutyrate, and then, if appropriate after concentration, the solution is applied, in a further process step, to synthetic silica, and in which the resultant product is, if appropriate, dried.

It would be desirable to provide a process for the production of 3-hydroxy-3-methylbutyrate which would be independent of inorganic production steps and which could be effected in living organisms thereby being environmentally sound and inexpensive. In this context, Lee et al. (Appl. Environ. Microbiol. 63 (1997), 4191-4195) describes a method for the production of 3-hydroxy-3-methylbutyrate by converting 3-methylbutyric acid to 3-hydroxy-3-methylbutyric acid using the microorganism Galactomyces reessii. However, although this process allowed the production of 3-hydroxy-3-methylbutyrate there is still a need to provide alternative efficient and cost effective ways of producing 3-hydroxy-3-methylbutyrate in particular by biological processes.

The present invention meets this demand for an alternative process for the production of 3-hydroxy-3-methylbutyrate and provides a method which is based on biological resources and allows to produce 3-hydroxy-3-methylbutyrate in vitro or in vivo in a microorganism and other species.

DETAILED DESCRIPTION OF THE INVENTION

Method for the Production of 3-hydroxy-3-methylbutyric Acid

In particular, the present invention relates to a method for the production of 3-hydroxy-3-methylbutyric acid (also referred to as beta-hydroxyisovalerate or HIV) from acetone and a compound which provides an activated acetyl group comprising the enzymatic conversion of acetone and a compound which provides an activated acetyl group into 3-hydroxy-3-methylbutyric acid.

Acetone is represented by the following formula: CH₃—(C═O)—CH₃. In a preferred embodiment the compound which provides an activated acetyl group is characterized by the following formula (I):

wherein X is selected from the group consisting of S—CH₂—CH₂—NH—CO—CH₂—CH₂—NH—CO—CH(OH)—C(CH₃)₂—CH₂—O—PO₂H—O—PO₂H—C₁₀H₁₃N₅O₇P (coenzyme A), S—CH₂—CH₂—NH—CO—CH₂—CH₂—NH—CO—CH(OH)—C(CH₃)₂—CH₂—O—PO₂H-polypeptide (acyl-carrier protein), S—CH₂—CH₂—NH—CO—CH₂—CH₂—NH—CO—CH(OH)—C(CH₃)₂—CH₂—OH (pantetheine), S—CH₂—CH₂—NH—CO—CH₃ (N-acetyl-cysteamine), S—CH₃ (methane thiol), S—CH₂—CH(NH₂)—CO₂H (cysteine), S—CH₂—CH₂—CH(NH₂)—CO₂H (homocysteine), S—CH₂—CH(NH—C₅H₈NO₃)—CO—NH—CH₂—CO₂H (glutathione), S—CH₂—CH₂—SO₃H (coenzyme M) and OH (acetic acid).

The conversion makes use of an enzyme which is capable of catalyzing the formation of a covalent bond between the carbon atom of the oxo (i.e. the C═O) group of acetone and the carbon atom (C²) corresponding to the methyl group of the compound which provides an activated acetyl group according to formula (I). According to this reaction scheme the oxo group of acetone reacts as an electrophile and the methyl group of the compound which provides an activated acetyl group according to formula (I) reacts as a nucleophile. The general reaction of the conversion of acetone and a compound which provides an activated acetyl group according to formula (I) is shown in FIG. 5.

The reaction can occur in one step, i.e. 3-hydroxy-3-methylbutyrate can be the direct product of a reaction catalyzed by the above described enzyme. Alternatively, the reaction may comprise two steps, in particular in the case where acetyl CoA is used as the compound which provides an activated acetyl group, in the sense that first an adduct of 3-hydroxy-3-methylbutyrate and the compound which provides an activated acetyl group is produced, e.g. 3-hydroxy-3-methylbutyryl-CoA, which is subsequently hydrolyzed, e.g. to 3-hydroxy-3-methylbutyrate and CoA. Thus, in the first alternative the enzyme catalyzes the complete reaction as shown in FIG. 5. In the second alternative, the enzyme catalyzes the formation of a covalent bond between the carbon atom of the oxo (i.e. the C═O) group of acetone and the carbon atom (C²) corresponding to the methyl group of the compound which provides an activated acetyl group but X stays in the molecule. X is then removed subsequently from the molecule by hydrolysis.

The present invention shows for the first time that it is possible to produce 3-hydroxy-3-methylbutyrate by making use of an enzyme which can transfer an activated acetyl group to acetone. In the prior art production of 3-hydroxy-3-methylbutyrate from isovaleric acid through bioconversion using the fungus Galactomyces reessii has been reported. However, considering that isovaleric acid is obtained from leucine through decarboxylation and that leucine itself derives in metabolism from the overall condensation of two molecules of pyruvate and one molecule of acetyl CoA, this production process is energetically unfavorable. The process of the present invention avoids this disadvantage.

In general, in the context of the present invention any enzyme could be used which accepts a compound which provides an activated acetyl group as defined above as one substrate as well as a substrate which contains as a component an acetone group. In one preferred embodiment, the enzyme is an enzyme which accepts acetyl CoA as a substrate. Examples for such enzymes are HMG CoA synthase, HMG CoA lyase or other C—C bond cleavage/condensation lyases. However, as will be explained below, also enzymes which normally use in the reaction that they catalyze in nature an acetyl-donor different from acetyl CoA, may use acetyl CoA or analogues thereof, e.g. the PksG protein.

In another preferred embodiment the enzyme is an enzyme which accepts as a substrate a compound which provides an activated acetyl group according to formula (I) in which X is an acyl-carrier-protein, such as the acetyl-S-AcpK protein encoded by the pksX gene cluster for producing bacillaene in Bacillus subtilis. An example for such an enzyme is the PksG protein. The PksG protein is one of the proteins encoded by the pksX gene cluster from Bacillus subtilis. The PksG protein is capable of catalyzing the transfer of a carboxymethyl group —CH₂—CO₂H from acetyl-S-AcpK to a β-ketothioester polyketide intermediate linked to one of the thiolation domains of the PksL protein, in a reaction which is analogous to that catalyzed by HMG CoA synthase. However, it has been shown in the context of the present invention that the PksG protein can also use acetyl CoA instead of the acetyl-S-AcpK protein as a donor of an activated acetyl group.

In one preferred embodiment the compound which provides an activated acetyl group is acetyl CoA. Acetyl CoA (also known as acetyl Coenzyme A) in chemical structure is the thioester between coenzyme A (a thiol) and acetic acid.

In another preferred embodiment the compound which provides an activated acetyl group has the formula (I) in which X is an acyl-carrier-protein, such as the acetyl-S-AcpK protein encoded by the pksX gene cluster for producing bacillaene in Bacillus subtilis.

Preferably, the enzyme employed in the process is an enzyme with the activity of a HMG CoA synthase (EC 2.3.3.10) and/or a PksG protein and/or an enzyme with the activity of a C—C bond cleavage/condensation lyase, such as a HMG CoA lyase (EC 4.1.3.4).

In one preferred embodiment, the method according to the present invention comprises the enzymatic conversion of acetone and acetyl CoA into 3-hydroxy-3-methylbutyrate with an enzyme which is capable of catalyzing the formation of a covalent bond between the carbon atom of the oxo (i.e. the C═O) group of acetone and the carbon atom C² of acetyl CoA according to formula (I).

In a preferred embodiment, the enzyme employed in the process according to the invention is an enzyme which has the activity of a HMG CoA synthase (EC 2.3.3.10) or an enzyme which has the activity of a PksG protein or an enzyme which has the activity of a C—C bond cleavage/condensation lyase, such as a HMG CoA lyase (EC 4.1.3.4).

In particular, it has been shown in the context of the present invention that HMG CoA synthase can accept acetone instead of its normal substrate acetoacetyl-CoA thereby allowing the conversion of acetyl-CoA (or a compound according to formula (I)) and acetone into 3-hydroxy-3-methylbutyrate.

Moreover, it has been shown in the context of the present invention that the PksG protein can use acetyl CoA as a substrate instead of the Ac-S-AcpK protein and can catalyze the reaction which is normally catalyzed by HMG CoA synthase. Thus, it is contemplated that also the PksG protein, which catalyzes a reaction analogous to the reaction of HMG CoA synthase, will be able to catalyze the conversion of acetone and a compound of formula (I) into 3-hydroxy-3-methylbutyrate. Moreover, it is contemplated that C—C bond cleavage/condensation lyases, such as HMG CoA lyase, can catalyze the conversion of acetyl-CoA and acetone into 3-hydroxy-3-methylbutyryl-CoA which in turn can be hydrolysed to 3-hydroxy-3-methylbutyrate and CoA.

HMG CoA Synthase

In the context of the present application the term “HMG CoA synthase” or “a protein/enzyme having the activity of a HMG CoA synthase” refers to any enzyme which is classified in the EC number EC 2.3.3.10 (formerly, HMG-CoA synthase has been classified as EC 4.1.3.5 but has been transferred to EC 2.3.3.10), in particular it refers to any enzyme which is able to catalyze the reaction where acetyl-CoA condenses with acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) (see FIG. 2) and the term also refers to any enzyme which is derived from such a HMG CoA synthase and which is capable of catalyzing the conversion of acetone and a compound which provides an activated acetyl group as defined above, preferably acetyl CoA, into 3-hydroxy-3-methylbutyrate.

The enzymatic activity of condensing acetyl-CoA with acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) can be measured by methods well known in the art. One possible and preferably used assay is described, e.g., in Clinkenbeard et al. (J. Biol. Chem. 250 (1975), 3108-3116). In this assay HMG-CoA synthase activity is measured by monitoring the decrease in absorbance at 303 nm that accompanies the acetyl-CoA-dependent disappearance of the enolate form of acetoacetyl-CoA. Preferably HMG CoA synthase activity is assayed as described in Example 3.

HMG CoA synthase is part of the mevalonate pathway. Two pathways have been identified for the synthesis of isopentenyl pyrophosphate (IPP), i.e. the mevalonate pathway and the glyceraldehyde 3-phosphate-pyruvate pathway. HMG CoA synthase catalyzes the biological Claisen condensation of acetyl-CoA with acetoacetyl-CoA and is a member of a superfamily of acyl-condensing enzymes that includes beta-ketothiolases, fatty acid synthases (beta-ketoacyl carrier protein synthase) and polyketide synthases.

HMG CoA synthase has been described for various organisms. Also amino acid and nucleic acid sequences encoding HMG CoA synthases from numerous sources are available. Generally, the sequences only share a low degree of overall sequence identity. For example, the enzymes from Staphylococcus or Streptococcus show only about 20% identity to those of human and avian HMG CoA synthase. In some sources it is reported that the bacterial HMG CoA synthases and their animal counterparts exhibit only about 10% overall sequence identity (Sutherlin et al., J. Bacteriol. 184 (2002), 4065-4070). However, the amino acid residues involved in the acetylation and condensation reactions are conserved among bacterial and eukaryotic HMG CoA synthases (Campobasso et al., J. Biol. Chem. 279 (2004), 44883-44888). The three-dimensional structure of three HMG CoA synthase enzymes has been determined and the amino acids crucial for the enzymatic reaction are in principle well characterized (Campobasso et al., loc. cit.; Chun et al., J. Biol. Chem. 275 (2000), 17946-17953; Nagegowda et al., Biochem. J. 383 (2004), 517-527; Hegardt, Biochem. J. 338 (1999), 569-582). In eukaryotes there exist two forms of the HMG CoA synthase, i.e. a cytosolic and a mitochondrial form. The cytosolic form plays a key role in the production of cholesterol and other isoprenoids and the mitochondrial form is involved in the production of ketone bodies.

In principle any HMG CoA synthase enzyme can be used in the context of the present invention, in particular from prokaryotic or eukaryotic organisms.

Prokaryotic HMG CoA synthases are described, e.g., from Staphylococcus aureus (Campobasso et al., loc. cit.; Uniprot accession number Q9FD87), Staphylococcus epidermidis (Uniprot accession number Q9FD76), Staphylococcus haemolyticus (Uniprot accession number Q9FD82), Enterococcus faecalis (Sutherlin et al., loc. cit.; Uniprot accession number Q9FD7), Enterococcus faecium (Uniprot accession number Q9FD66), Streptococcus pneumonia (Uniprot accession number Q9FD56), Streptococcus pyogenes (Uniprot accession number Q9FD61) and Methanobacterium thermoautotrophicum (accession number AE000857), Borrelia burgdorferi (NCBI accession number BB0683).

Moreover, the following Table A lists some known HMG CoA synthases from prokaryotes:

TABLE A Swissprot/TrEmbl Accession number Organism Q9YAS0 Aeropyrum pernix A7Z4Y2 Bacillus amyloliquefaciens P40830|2874037340 Bacillus subtilis B8G795 Chloroflexus aggregans A5EUV4 Dichelobacter nodosus A5FM54 Flavobacterium johnsoniae Q18GC4 Haloquadratum walsbyi B9LS15 Halorubrum lacusprofundi A9B8F0 Herpetosiphon aurantiacus A2BMY8 Hyperthermus butylicus Q5FLB7 Lactobacillus acidophilus Q03QR0 Lactobacillus brevis Q1GAH5 Lactobacillus delbrueckii B2GBL1 Lactobacillus fermentum B1MZ51 Leuconostoc citreum Q03WZ0 Leuconostoc mesenteroides A4YH99 Metallosphaera sedula A5UNI8 Methanobrevibacter smithii Q58941 Methanocaldococcus jannaschii Q12UR3 Methanococcoides burtonii A6USZ1 Methanococcus aeolicus A4FWW6 Methanococcus maripaludis A6UPL1 Methanosarcina mazei A2STY2 Methanocorpusculum labreanum Q8TVL0 Methanopyrus_andleri Q8PYJ0 Methanosarcina mazei Q2NHU7 Methanosphaera stadtmanae Q2FPH4 Methanospirillum hungatei B2HGT6 Mycobacterium marinum Q3IMZ7 Natronomonas pharaonis Q8EP69 Oceanobacillus iheyensis Q04F95 Oenococcus oeni Q03FU5 Pediococcus pentosaceus Q6L233 Picrophilus torridus A6G7N7 Plesiocystis pacifica A4WJ12 Pyrobaculum arsenaticum A7NHZ7 Roseiflexus castenholzii Q8CN06 Staphylococcus epidermidis Q4L958 Staphylococcus haemolyticus Q4A0D6 Staphylococcus saprophyticus B4U364 Streptococcus equi Q8DUI5 Streptococcus mutans Q4J933 Sulfolobus acidocaldarius Q971K8 Sulfolobus tokodaii Q9HI87 Thermoplasma acidophilum Q31EW2 Thiomicrospira crunogena Q51798 Pyrococcus furiosus A5VJB7 Lactobacillus reuteri Q7CF79 Streptococcus pyogenes Q9UWU0 Sulfolobus solfataricus

Eukaryotic HMG CoA synthases are described, e.g., from fungi, such as Schizosaccharomyces pombe (accession numbers U32187 and P54874), Saccharomyces cerevisiae (accession number P54839), plants, such as Arabidopsis thaliana (accession numbers X83882 and P54873), Pinus sylvestris (accession number X96386) and animals, such as Caenorhabditis elegans (accession number P54871), Mus musculus (mitochondrial; accession number P54869 and Hegardt, Biochem. J. 338 (1999), 569-582), Rattus norvegicus (mitochondrial: accession number P22791 and Hegardt, Biochem. J. 338 (1999); cytosolic: accession number P17425), 569-582), Chinese hamster (Cricetulus griseus: accession number P13704), Sus scrofa (mitochondrial; accession number U90884 and Hegardt, Biochem. J. 338 (1999), 569-582), Homo sapiens (mitochondrial: accession number P54868 and Hegardt, Biochem. J. 338 (1999), 569-582; cytosolic: accession number Q01581), Blattella germanica (cytosolic form 1; accession number P54961), Blattella germanica (cytosolic form 2; accession number P54870) and Gallus gallus (cytosolic; accession number P23228).

Examples of HMG CoA synthases from different organisms are given in SEQ ID NO: 1 to 14. SEQ ID NO: 1 shows the sequence of the cytoplasmic HMG CoA synthase of Caenorhabditis elegans (P54871, gene bank F25B4.6), SEQ ID NO: 2 shows the sequence of the cytoplasmic HMG CoA synthase of Schizosaccharomyces pombe (fission yeast; P54874), SEQ ID NO: 3 shows the sequence of the cytoplasmic HMG CoA synthase of Saccharomyces cerevisiae (baker's yeast; P54839, gene bank CAA65437.1), SEQ ID NO: 4 shows the sequence of the cytoplasmic HMG CoA synthase of Arabidopsis thaliana (Mouse-ear cress; P54873), SEQ ID NO: 5 shows the sequence of the cytoplasmic HMG CoA synthase of Dictyostelium discoideum (Slime mold; P54872, gene bank L2114), SEQ ID NO: 6 shows the sequence of the cytoplasmic HMG CoA synthase of Blattella germanica (German cockroach; P54961, gene bank X73679), SEQ ID NO: 7 shows the sequence of the cytoplasmic HMG CoA synthase of Gallus gallus (Chicken; P23228, gene bank CHKHMGCOAS), SEQ ID NO: 8 shows the sequence of the cytoplasmic HMG CoA synthase of Homo sapiens (Human; Q01581, gene bank X66435), SEQ ID NO: 9 shows the sequence of the mitochondrial HMG CoA synthase of Homo sapiens (Human; P54868, gene bank X83618), SEQ ID NO: 10 shows the sequence of the mitochondrial HMG CoA synthase of Dictyostelium discoideum (Slime mold; Q86HL5, gene bank XM_(—)638984), SEQ ID NO: 11 shows the sequence of the HMG CoA synthase of Staphylococcus epidermidis (Q9FD76), SEQ ID NO: 12 shows the sequence of the HMG CoA synthase of Lactobacillus fermentum(B2 GBL1), SEQ ID NO: 13 shows the sequence of the HMG CoA synthase of Hyperthermus butylicus (A2BMY8), SEQ ID NO: 14 shows the sequence of the HMG CoA synthase of Chloroflexus aggregans (B8G795), SEQ ID NO: 24 shows the sequence of the HMG CoA synthase of Lactobacillus delbrueckii (Q1GAH5) and SEQ ID NO: 25 shows the sequence of the HMG CoA synthase of Staphylococcus haemolyticus Q4L958 (I98>V difference compared to wild type protein).

In a preferred embodiment of the present invention the HMG CoA synthase is an enzyme comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 14 or a sequence which is at least n % identical to any of SEQ ID NOs: 1 to 14 and having the activity of a HMG CoA synthase with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99.

Preferably, the degree of identity is determined by comparing the respective sequence with the amino acid sequence of any one of the above-mentioned SEQ ID NOs. When the sequences which are compared do not have the same length, the degree of identity preferably either refers to the percentage of amino acid residues in the shorter sequence which are identical to amino acid residues in the longer sequence or to the percentage of amino acid residues in the longer sequence which are identical to amino acid residues in the shorter sequence. The degree of sequence identity can be determined according to methods well known in the art using preferably suitable computer algorithms such as CLUSTAL.

When using the Clustal analysis method to determine whether a particular sequence is, for instance, 80% identical to a reference sequence default settings may be used or the settings are preferably as follows: Matrix: blosum 30; Open gap penalty: 10.0; Extend gap penalty: 0.05; Delay divergent: 40; Gap separation distance: 8 for comparisons of amino acid sequences. For nucleotide sequence comparisons, the Extend gap penalty is preferably set to 5.0.

Preferably, the degree of identity is calculated over the complete length of the sequence.

The HMG CoA synthase employed in the process according to the invention can be a naturally occurring HMG CoA synthase or it can be a HMG CoA synthase which is derived from a naturally occurring HMG CoA synthase, e.g. by the introduction of mutations or other alterations which, e.g., alter or improve the enzymatic activity, the stability, etc.

The term “HMG CoA synthase” or “a protein/enzyme having the activity of a HMG CoA synthase” in the context of the present application also covers enzymes which are derived from a HMG CoA synthase, which are capable of producing 3-hydroxy-3-methylbutyrate by an enzymatic conversion of acetone and a compound which provides an activated acetyl group as defined above, preferably acetyl-CoA, but which only have a low affinity to acetoacetyl-CoA as a substrate or do no longer accept acetoacetyl-CoA as a substrate. Such a modification of the preferred substrate of a HMG CoA synthase allows to improve the conversion of acetone into 3-hydroxy-3-methylbutyrate and to reduce the production of the by-product, e.g. HMG-CoA. Methods for modifying and/or improving the desired enzymatic activities of proteins are well-known to the person skilled in the art and include, e.g., random mutagenesis or site-directed mutagenesis and subsequent selection of enzymes having the desired properties or approaches of the so-called “directed evolution”.

For example, for genetic engineering in prokaryotic cells, a nucleic acid molecule encoding HMG CoA synthase can be introduced into plasmids which permit mutagenesis or sequence modification by recombination of DNA sequences. Standard methods (see Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, N.Y., USA) allow base exchanges to be performed or natural or synthetic sequences to be added. DNA fragments can be connected to each other by applying adapters and linkers to the fragments. Moreover, engineering measures which provide suitable restriction sites or remove surplus DNA or restriction sites can be used. In those cases, in which insertions, deletions or substitutions are possible, in vitro mutagenesis, “primer repair”, restriction or ligation can be used. In general, a sequence analysis, restriction analysis and other methods of biochemistry and molecular biology are carried out as analysis methods. The resulting HMG CoA synthase variants are then tested for their enzymatic activity and in particular for their capacity to prefer acetone as a substrate rather than acetoacetylCoA. An assay for measuring the capacity of a HMG CoA synthase to use acetone as a substrate is described in Example 5. The formation of 3-hydroxy-3-methylbutyrate can be detected by comparison with standard compound, e.g. after separation by thin-layer chromatography, LC/MS and colorimetric assay after its derivatization or by mass spectrometry.

In particular, a reaction is carried out in a reaction mixture containing 40 mM Tris-HCl pH 8, 5 to 50 mM acetyl-CoA, 100 to 500 mM acetone, 1 MgCl₂ (except for mitochondria HMG-CoA synthase), 0.5 mM DTT (dithiothreitol) and enzyme varying in the range from 0.2 to 8 mg/ml. Control reactions are carried in the absence of enzyme and one of the substrates.

The progress of synthesis is followed by analyzing aliquots taken after increasing period of incubation at 30 or 37° C. Typically, an aliquot of 50 μl is removed after 48 h of incubation, heated for 1 min at 100° C. to eliminate the proteins, centrifuged and the supernatant is transferred to a clean vial for HIV detection by mass spectrometry. A solution of 3-hydroxy-3-methylbutyrate is prepared in 40 mM Tris-HCl pH 8, 1 mM MgCl₂, 0.5 mM DTT, heated as described above and used as reference.

The samples are analyzed on a PE SCIEX® API 2000 triple quadrupole mass spectrometer (mass spectrometer, Perkin-Elmer) in negative ion mode with H₂O/acetonitrile=60/40 containing 0.1% triethylamine as mobile phase, flow rate was 40 μl/min. 10 μl of each supernatant are mixed with an equal quantity of mobile phase and directly injected into the mass spectrometer. The presence of [3-hydroxy-3-methylbutyrate-H]⁻ ion is monitored.

3-hydroxy-3-methylbutyrate synthesis can also be carried out in the presence of radiolabeled [2-¹⁴C] acetone. The formation of product is analyzed after separation of the reaction mixture by TLC or HPLC.

In a preferred embodiment the HMG CoA synthase employed in the present invention is an enzyme which has a K_(M) value for acetone of 300 mM or lower, preferably of 250 mM or lower even more preferably of 200 mM or lower and particularly preferred of 150 mM or lower. It is preferred that the K_(M) value is determined under the conditions described in Example 7. In another preferred embodiment the HMG CoA synthase employed in the present invention has a k_(cat) value for the described reaction of at least 0.1×10⁻⁴ sec⁻¹, preferably at least 0.2×10⁻⁴ sec⁻¹, even more preferably at least 0.5×10⁻⁴ sec⁻¹ and particularly preferred at least 1×10⁻⁴ sec⁻¹, at least 2×10⁻⁴ sec⁻¹, at least 3×10⁻⁴ sec⁻¹ or at least 5×10⁻⁴ sec⁻¹. It is preferred that the k_(cat) value is determined under the conditions described in Example 7

It is known in the art that His264 of avian HMG CoA synthase plays a role in the interaction of the enzyme with acetoacetyl-CoA and that the Ala264 variant lacks interaction with the oxygen of the thioester moiety of acetoacetyl-CoA (Misraa et al., Biochem. 35 (1996), 9610-9616). Thus, in order to develop variants of HMG CoA synthase which show a lower acceptance of acetoacetyl-CoA as a substrate but which accept acetone as a substrate, it is conceivable to systematically mutate in a HMG CoA synthase the histidine residue which corresponds to His264 of the avian HMG CoA synthase described in Misraa et al. (loc. cit.) so as to reduce or disable the acceptance of acetoacetyl-CoA as substrate.

In addition, HMG CoA synthase variants can be provided which show an increased activity. Steussy et al. (Biochemistry 45 (2006), 14407-14414), for example, describe a mutant of the Enterococcus faecalis HMG CoA synthase in which Ala110 was changed to Gly110 and which shows an 140-fold increase of the overall reaction rate.

Methods for identifying variants with improved enzymatic properties as regards the production of 3-hydroxy-3-methylbutyrate may also be carried out in the presence of a cofactor which allows for a steric and/or electronic complementation in the catalytic site of the enzyme/enzymes due to the fact that the substrate acetone is shorter than the natural substrate acetoacetyl-CoA of, HMG CoA synthase. One example of such a cofactor would be coenzyme A or a structurally closely related molecule such as S-nitroso-CoA.

The modified version of the HMG CoA synthase accepting acetone as a substrate but having a low affinity to acetoacetyl-CoA as a substrate or no longer accepting acetoacetyl-CoA as a substrate may be derived from a naturally occurring HMG CoA synthase or from an already modified, optimized or synthetically synthesized HMG CoA synthase.

PksG Protein

Another example for a protein which can be used in a method according to the invention is a PksG protein. In the context of the present application the term “PksG protein” or “a protein/enzyme having the activity of a PksG protein” refers to any enzyme which is able to catalyze the reaction which is naturally catalyzed by the PksG protein, i.e the transfer of —CH₂COO⁻ from acetyl-S-AcpK (Ac-S-AcpK) to a β-ketothioester polyketide intermediate linked to one of the thiolation domains of the PksL protein. This is a reaction which is analogous to that catalyzed by HMG CoA synthase with the difference that the acetyl-thioester of the phosphopantetheyl moiety is attached to a carrier protein rather than to part of Coenzyme A. Although the PksG protein in the reaction which it naturally catalyzes transfers the acetyl group from acetyl-S-AcpK to an acceptor, it has been shown in the context of the present invention that the PksG protein can also effect the reaction which is normally catalyzed by HMG CoA synthase, i.e. the synthesis of HMG CoA starting from acetoacetyl CoA and acetyl CoA (see Example 3 where it is shown in Table 1 that the enzyme from Mycobacterium marinum (B2HGT6) can act on acetoacetyl CoA and acetyl CoA).

The enzymatic activity of the PksG protein can be measured by methods known in the art. One possible and preferably used assay is described, e.g., in Calderone et al. (Proc. Natl. Acad. Sci. USA 103 (2006), 8977-8982). In this assay acetoacetyl (Acac)-S-PksL-T2 is used as a model substrate and is incubated together with Ac-S-AcpK and the PksG protein. The formation of HMG-S-PksL-T2 indicates that the PksG protein is capable of transferring the carboxymethyl group —CH₂—CO₂H from Ac-S-AcpK to (Acac)-S-PksL-T2. The formation of HMG-S-PksL-T2 can be determined either by electrospray ionization (ESI)-FTMS or in an autoradiography. In a preferred embodiment the corresponding assays are carried out as described on page 8982 of Calderone et al. (Proc. Natl. Acad. Sci. USA 103 (2006), 8977-8982).

The PksG protein is part of the pksX pathway in Bacillus subtilis which encodes the enzymes responsible for the biosynthesis of bacillaene (Butcher et al., Proc. Natl. Acad. Sci. USA 104 (2007), 1506-1509). The encoded proteins are AcpK, PksC, PksL, PksF, PksG, PksH and PksI. According to Calderone et al. (Proc. Natl. Acad. Sci. USA 103 (2006), 8977-8982) these enzymes act to incorporate an acetate derived β-methyl branch on an acetoacetyl-S-carrier protein.

In a preferred embodiment of the present invention the PksG protein is an enzyme comprising an amino acid sequence as shown in SEQ ID NO: 15 or 16 or a sequence which is at least n % identical to SEQ ID NO: 15 or 16 and having the activity of a PksG protein with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99.

SEQ ID NO: 15 shows the amino acid sequence of the PksG protein of Bacillus subtilis (P40830) and SEQ ID NO: 16 shows the amino acid sequence of the PksG protein of Mycobacterium marinum (B2HGT6).

As regards the determination of the degree of sequence identity the same applies as has been set forth above in connection with HMG CoA synthase.

The PksG protein employed in the process according to the invention can be a naturally occurring PksG protein or it can be a PksG protein which is derived from a naturally occurring PksG protein, e.g. by the introduction of mutations or other alterations which, e.g., alter or improve the enzymatic activity, the stability, etc.

The term “PksG protein” or “a protein/enzyme having the activity of a PksG protein” in the context of the present application also covers enzymes which are derived from a PksG protein, which are capable of producing 3-hydroxy-3-methylbutyrate by an enzymatic conversion of acetone and a compound which provides an activated acetyl group as defined above, preferably acetyl-CoA, but which only have a low affinity to their natural substrate or do no longer accept their natural substrate. Such a modification of the preferred substrate of a PksG protein allows to improve the conversion of acetone into 3-hydroxy-3-methylbutyrate and to reduce the production of unwanted by-product. Methods for modifying and/or improving the desired enzymatic activities of proteins are well-known to the person skilled in the art and have been described above. The resulting PksG protein variants are then tested for their enzymatic activity and in particular for their capacity to prefer acetone as a substrate. An assay for measuring the capacity of a PksG protein to use acetone as a substrate is the one described in Example 5 for HMG-CoA synthase. The formation of 3-hydroxy-3-methylbutyrate can be detected as described above.

Such methods for identifying variants with improved enzymatic properties as regards the production of 3-hydroxy-3-methylbutyrate may also be carried out in the presence of a cofactor which allows for a steric and/or electronic complementation in the catalytic site of the enzyme/enzymes due to the fact that the substrate acetone is shorter than the natural substrate of the PksG protein.

The modified version of the PksG protein accepting acetone as a substrate but having a low affinity to or no longer accepting its natural substrate may be derived from a naturally occurring PksG protein or from an already modified, optimized or synthetically synthesized PksG protein.

C—C Bond Cleavage/Condensation Lyase, HMG CoA Lyase

In the context of the present invention the term “C—C bond cleavage/condensation lyase” or “a protein/enzyme having the activity of a C—C bond cleavage/condensation lyase” refers to an enzyme which is capable of cleaving or forming by condensation a C—C bond and which contains a so-called TIM (triose-phosphate isomerase) barrel domain. This TIM barrel domain is found in a number of pyruvate binding enzymes and acetyl-CoA dependent enzymes (Forouhar et al. J. Biol. Chem. 281 (2006), 7533-7545). The TIM barrel domain has the classification lineage 3.20.20.150 in the CATH protein classification database (www.cathdb.info/cathnode/3.20.20.150).

The term “C—C bond cleavage/condensation lyases” in particular includes enzymes which are classified as isopropylmalate synthase (EC 2.3.3.13), as homocitrate synthase (EC 2.3.3.14) or as 4-hydroxy-2-ketovalerate aldolase (EC 4.1.3.39). Isopropylmalate synthase catalyzes the following reaction: acetyl-CoA+3-methyl-2-oxobutanoate+H₂O

(2S)-2-isopropylmalate+CoA. Examples for such enzymes are the corresponding enzyme from Brucella abortus (strain 2308; Q2YRT1) and the corresponding enzyme from Hahella chejuensis (strain KCTC 2396; Q2SFA7).

A homocitrate synthase (EC 2.3.3.14) is an enzyme that catalyzes the chemical reaction acetyl-CoA+H₂O+2-oxoglutarate

(R)-2-hydroxybutane-1,2,4-tricarboxylate+CoA. The 4-hydroxy-2-ketovalerate aldolase catalyzes the chemical reaction 4-hydroxy-2-oxopentanoate

acetaldehyde+pyruvate.

In the context of the present invention the term “HMG CoA lyase” or “a protein/enzyme having the activity of a HMG CoA lyase” refers to any enzyme which is classified in the EC number EC 4.1.3.4, in particular it refers to any enzyme which is able to catalyze the cleavage of HMG CoA into acetyl CoA and acetoacetate (see FIG. 3) or the reverse of this reaction, i.e. the production of HMG CoA through the condensation of acetyl CoA and acetoacetate, and the term also refers to any enzyme which is derived from such a HMG CoA lyase and which is capable of catalyzing the conversion of acetone and a compound providing an activated acetyl group as defined above, preferably acetyl CoA, into 3-hydroxy-3-methylbutyryl-CoA. In the context of the present invention the produced 3-hydroxy-3-methylbutyryl-CoA can then be hydrolyzed to produce 3-hydroxy-3-methylbutyrate. This could be achieved by measures known to the person skilled in the art, e.g. by making use of an acyl-CoA hydrolase (EC 3.1.2.20) or an acyl-CoA transferase (EC 2.8.3.8).

The enzymatic activity of HMG CoA lyase can be measured by methods well known in the art. One possible assay is described, e.g., in Mellanby et al. (Methods of Enzymatic Analysis; Bergmeyer Ed. (1963), 454-458). In particular, the enzyme activity is measured by a spectrophotometric assay using the NADH-dependent reduction of acetoacetate by 3-hydroxybutyrate dehydrogenase.

Preferably HMG CoA lyase activity is assayed as described in Example 4. In such an assay the reaction mixture (1 ml) contains 40 mM Tris-HCl pH 8, 1 mM MgCl₂, 0.5 mM DTT, 0.4 mM HMG-CoA, 0.2 mM NADH, 5 units of 3-hydroxybutyrate dehydrogenase and is incubated for 5 min before adding 0.005 mg/ml of HMG-CoA lyase and then the progress of the reaction is monitored by the decrease in absorbance at 340 nm.

The reaction catalyzed by HMG CoA lyase is described in some instances to require the presence of a divalent cation, such as Mg²⁺ or Mn²⁺. Thus, it is preferred that an assay for determining the activity of HMG CoA lyase includes such divalent cations and that the method according to the invention for the production of 3-hydroxy-3-methylbutyric acid, if it makes use of HMG CoA lyase, is carried out in the presence of such cations.

HMG CoA lyase is part of the hepatic ketogenesis. It catalyses the terminal reaction in the hepatic ketogenesis which is a key step of this pathway. The reaction is also an important step in leucine catabolism.

HMG CoA lyase has been described for various organisms. Amino acid and nucleic acid sequences encoding HMG CoA lyases are available from numerous sources. Generally, the sequences only share an intermediate degree of overall sequence identity. For example, the enzymes from Bacillus subtilis or Brucella melitensis show only about 45% identity to those of human HMG CoA lyase (Forouhar et al., J. Biol. Chem. 281 (2006), 7533-7545). The three-dimensional structure of various HMG CoA lyase enzymes has been determined and the amino acids crucial for the enzymatic reaction are in principle well characterized (Forouhar et al., loc. cit.; Fu et al., J. Biol. Chem. 281 (2006), 7526-7532). In eukaryotes the HMG CoA lyase is located in the mitochondrial matrix.

In principle any HMG CoA lyase enzyme can be used in the context of the present invention, in particular from prokaryotic or eukaryotic organisms.

Prokaryotic HMG CoA lyases are described, e.g., from Brucella abortus (UniProt accession numbers Q2YPL0 and B2S7S2), Bacillus subtilis (UniProt accession number 034873), Bacillus licheniformis (Fu et al., loc. cit.), Pseudomonas syringae (UniProt accession numbers Q4ZTL2 and Q4ZRW6), Pseudomonas mevalonii (UniProt accession number P13703), Shewanella piezotolerans (UniProt accession number B8CRY9), Cellvibrio japonicus (UniProt accession number B3PCQ7), Azotobacter vinelandii (UniProt accession numbers C1DJK8 and C1DL53), Herminiimonas arsenicoxydans (UniProt accession number A4G1F2) and Burkholderia cenocepacia (UniProt accession number A2VUW7).

Moreover, the following Table B lists some known HMG CoA lyases from prokaryotes:

TABLE B Swissprot/TrEmbl Accession number Organism Q6MHG9 Bdellovibrio bacteriovorus A2TNG9 Dokdonia donghaensis Q0C392 Hyphomonas neptunium B2HGF8 Mycobacterium marinum Q0K3L2 Ralstonia eutropha A9IB40 Bordetella petrii Q0B1Z9 Burkholderia ambifaria A5FHS2 Flavobacterium johnsoniae Q5X487 Legionella pneumophila A1VJH1 Polaromonas naphthalenivorans Q5WKL8 Bacillus clausii A9IFQ7 Bordetella petrii A6H0L4 Flavobacterium psychrophilum Q8F7U7 Leptospira interrogans A1VLB1 Polaromonas naphthalenivorans A9IR28 Bordetella petrii B1HZX7 Lysinibacillus sphaericus A1VT25 Polaromonas naphthalenivorans Q9KDS7 Bacillus halodurans A9HXH6 Bordetella petrii Q39QG8 Geobacter metallireducens Q2GBZ7 Novosphingobium aromaticivorans Q0KC96 Ralstonia eutropha Q7CSK6 Agrobacterium tumefaciens Q65IT6 Bacillus licheniformis Q7NX69 Chromobacterium violaceum B9LMV8 Halorubrum lacusprofundi A6F2L0 Marinobacter algicola Q8ERF9 Oceanobacillus iheyensis Q88HG4 Pseudomonas putida Q0KF83 Ralstonia eutropha Q0VL35 Alcanivorax borkumensis B2JST8 Burkholderia phymatum A9AXJ6 Herpetosiphon aurantiacus B1ML74 Mycobacterium abscessus Q88H25 Pseudomonas putida Q11V59 Cytophaga hutchinsonii Q0BWU6 Hyphomonas neptunium A1BBP4 Paracoccus denitrificans Q3IGB2 Pseudoalteromonas haloplanktis Q21QR6 Rhodoferax ferrireducens Q21RT0 Rhodoferax ferrireducens A4CMM6 Robiginitalea biformata A7NGX6 Roseiflexus castenholzii A7NPP8 Roseiflexus castenholzii A7NPR9 Roseiflexus castenholzii Q163P7 Roseobacter denitrificans A4X0W1 Salinispora tropica A9KVP4 Shewanella baltica Q12LZ6 Shewanella denitrificans A8FT92 Shewanella sediminis Q82CR7 Streptomyces avermitilis Q72IH0 Thermus thermophilus A9WGE2 Chloroflexus aurantiacus B7H4C6 Acinetobacter baumannii

Eukaryotic HMG CoA lyases are described, e.g., from plants, such as radish (Raphanus sativus) and Zea mays (Accession number B6U7B9, gene bank ACG45252) and animals, such as human (Homo sapiens; UniProt accession number P35914), Cynomolgus monkey (UniProt accession number Q8XZ6), Sumatran orangutan (Pongo abelii; UniProt accession number Q5R9E1), rat (Rattus norvegicus; UniProt accession number P97519; Fu et al., loc. cit.), Mus musculus (UniProt accession number P38060), duck (Anas spec.), cattle (Bos taurus; UniProt accession number Q29448), goat (Capra hircus), pigeon (Columba livia), chicken (Gallus gallus; UniProt accession number P35915), sheep (Ovis aries), pig (Sus scrofa), Danio rerio (Brachydanio rerio; A8WG57, gene bank BC154587) and from the protozoa Tetrahymena pyriformis.

Examples of HMG CoA lyases from different organisms are given in SEQ ID NOs: 17 to 23. SEQ ID NO: 17 shows the sequence of the HMG CoA lyase of Zea mays (Accession number B6U7B9, gene bank ACG45252), SEQ ID NO: 18 shows the sequence of the HMG CoA lyase of Danio rerio (Brachydanio rerio; A8WG57, gene bank BC154587), SEQ ID NO: 19 shows the sequence of the HMG CoA lyase of Bos taurus (Uniprot accession number Q29448) and SEQ ID NO: 20 shows the sequence of the HMG CoA lyase of Homo sapiens (mitochondrial, Uniprot accession number P35914, gene bank HUMHYMEGLA), SEQ ID NO: 21 shows the sequence of the HMG CoA lyase of Pseudomonas putida (Q88H25), SEQ ID NO: 22 shows the sequence of the HMG CoA lyase of Acinetobacter baumannii (B7H4C6) and SEQ ID NO: 23 shows the sequence of the HMG CoA lyase of Thermus thermophilus (Q72IH0).

In a preferred embodiment of the present invention the HMG CoA lyase is an enzyme comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 to 23 or a sequence which is at least n % identical to any of SEQ ID NOs: 17 to 23 and having the activity of a HMG CoA lyase with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99.

As regards the determination of the degree of sequence identity the same applies as has been set forth above in connection with HMG CoA synthase.

The HMG CoA lyase employed in the process according to the invention can be a naturally occurring HMG CoA lyase or it can be a HMG CoA lyase which is derived from a naturally occurring HMG CoA lyase, e.g. by the introduction of mutations or other alterations which, e.g., alter or improve the enzymatic activity, the stability, etc.

The term “HMG CoA lyase” or “a protein/enzyme having the activity of a HMG CoA lyase” in the context of the present application also covers enzymes which are derived from a HMG CoA lyase, which are capable of producing 3-hydroxy-3-methylbutyryl-CoA by a condensation of acetone and a compound which provides an activated acetyl group as defined above, preferably acetyl-CoA but which only have a low affinity to acetoacetate as a substrate or do no longer accept acetoacetate as a substrate. Such a modification of the preferred substrate of a HMG CoA lyase allows to improve the conversion of acetone into 3-hydroxy-3-methylbutyryl-CoA and to reduce the production of the by-product HMG-CoA. Methods for modifying and/or improving the desired enzymatic activities of proteins are well-known to the person skilled in the art and have been described above.

The capacity of a given enzyme to catalyze the production of 3-hydroxy-3-methylbutyryl-CoA can be determined in an assay as described in Example 6.

The modified version of the HMG CoA lyase accepting acetone as a substrate but having a low affinity to acetoacetate as a substrate or no longer accepting acetoacetate as a substrate may be derived from a naturally occurring HMG CoA lyase or from an already modified, optimized or synthetically synthesized HMG CoA lyase.

Reactions May Conducted in Cellulo or in vitro

In the process according to the invention it is possible to employ only one enzyme as defined above, e.g. only a HMG CoA synthase or only a HMG CoA lyase or only a PksG protein. However, it is of course also possible to employ more than one activity, i.e. different enzymes, in particular any combination of a HMG CoA synthase and a HMG CoA lyase and a PksG protein. E.g., in the case of an in vitro method, more than one enzyme activity can be added to the reaction mixture, either simultaneously or subsequently in any possible order. In an in vivo method employing organisms, in particular microorganisms, it is, e.g., possible to use an organism, in particular microorganism, expressing an enzyme as defined above. However, it is also conceivable to use an organism/microorganism expressing any possible combination of the above mentioned enzymes. Moreover, it is also possible to use a mixture of two or more types of organisms/microorganisms with one type expressing one enzyme and another expressing another enzyme. These different types can then be cocultivated.

The enzyme, e.g. the HMG CoA synthase and/or PksG protein and/or a C—C bond cleavage/condensation lyase, such as a HMG CoA lyase, employed in the process according to the present invention can be a natural version of the protein or a synthetic protein as well as a protein which has been chemically synthesized or produced in a biological system or by recombinant processes. The enzyme, e.g. the HMG CoA synthase and/or PksG protein and/or a C—C bond cleavage/condensation lyase, such as a HMG CoA lyase, may also be chemically modified, for example in order to improve its/their stability, resistance, e.g. to temperature, for facilitating its/their purification or its immobilization on a support. The enzyme/enzymes may be used in isolated form, purified form, in immobilized form, as a crude or partially purified extract obtained from cells synthesizing the enzyme/enzymes, as chemically synthesized enzyme(s), as recombinantly produced enzyme(s), in the form of microorganisms producing them etc.

The process according to the present invention may be carried out in vitro or in vivo. An in vitro reaction is understood to be a reaction in which no cells are employed, i.e. an acellular reaction.

For carrying out the process in vitro the substrates for the reaction and the enzyme/enzymes are incubated under conditions (buffer, temperature, cofactors etc.) allowing the enzyme/enzymes to be active and the enzymatic conversion to occur. The reaction is allowed to proceed for a time sufficient to produce 3-hydroxy-3-methylbutyrate. The production of 3-hydroxy-3-methylbutyrate and/or 3-hydroxy-3-methylbutyryl-CoA can be detected by comparison with standard compound after separation by thin-layer chromatography, LC/MS and colorimetric assay after its derivatization.

The enzyme/enzymes may be in any suitable form allowing the enzymatic reaction to take place. It/they may be purified or partially purified or in the form of crude cellular extracts or partially purified extracts. It is also possible that the enzyme/enzymes is immobilized on a suitable carrier.

Since the substrate acetone is in general shorter than the natural substrate used by the enzyme, e.g. acetoacetyl-CoA/acetoacetate used by HMG CoA synthase and HMG CoA lyase, respectively, it may be advantageous to add to the reaction mixture a cofactor which allows for a steric and/or electronic complementation in the catalytic site of the enzyme/enzymes. One example of such a cofactor, in the case of HMG CoA synthase, would be coenzyme A or a structurally closely related molecule such as S-nitroso-CoA.

For carrying out the process in vivo use is made of a suitable organism/microorganism(s) which is/are capable of providing the substrates, i.e. acetone and a compound which provides an activated acetyl group as defined above, and an enzyme which is capable of catalyzing the formation of a covalent bond between the carbon atom of the oxo (i.e. the C═O) group of acetone and the carbon atom (C²) corresponding to the methyl group of the compound which provides an activated acetyl group. In a preferred embodiment said enzyme is a HMG CoA synthase and/or PksG protein and/or a C—C bond cleavage/condensation lyase, such as a HMG CoA lyase.

Recombinant Microorganisms

Thus, in the case of this embodiment the method according to the invention is characterised in that the conversion of acetone and a compound which provides an activated acetyl group is realized in the presence of an organism, preferably a microorganism capable of producing acetone and expressing an enzyme which is capable of the formation of a covalent bond between the carbon atom of the oxo (i.e. the C═O) group of acetone and the carbon atom (C²) corresponding to the methyl group of the compound which provides an activated acetyl group, preferably expressing an enzyme with the activity of a HMG CoA synthase (EC 2.3.3.10) and/or expressing a PksG protein and/or expressing an enzyme with the activity of a C—C bond cleavage/condensation lyase, such as a HMG CoA lyase (EC 4.1.3.4).

The term “which is capable of producing acetone” in the context of the present invention means that the organism/microorganism has the capacity to produce acetone within the cell due to the presence of enzymes providing enzymatic activities allowing the production of acetone from metabolic precursors.

Acetone is produced by certain microorganisms, such as Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium cellulolyticum, Bacillus polymyxa and Pseudomonas putida. The synthesis of acetone is best characterized in Clostridium acetobutylicum. It starts out with a reaction (reaction step 1) in which two molecules of acetyl-CoA are condensed into acetoacetyl-CoA. This reaction is catalyzed by acetyl-CoA acetyltransferase (EC 2.3.1.9). Acetoacetyl-CoA is then converted into acetoacetate by a reaction with acetic acid or butyric acid resulting also in the production of acetyl-CoA or butyryl-CoA (reaction step 2). This reaction is catalyzed e.g. by acetoacetylCoA transferase (EC 2.8.3.8). AcetoacetylCoA transferase is known from various organisms, e.g. from E. coli in which it is encoded by the atoAD gene or from Clostridium acetobutylicum in which it is encoded by the ctfAB gene. However, also other enzymes can catalyze this reaction, e.g. 3-oxoacid CoA transferase (EC 2.8.3.5) or succinate CoA ligase (EC 6.2.1.5).

Finally, acetoacetate is converted into acetone by a decarboxylation step (reaction step 3) catalyzed by acetoacetate decarboxylase (EC 4.1.1.4).

The above described reaction steps 1 and 2 and the enzymes catalyzing them are not characteristic for the acetone synthesis and can be found in various organism. In contrast, reaction step 3 which is catalyzed by acetoacetate decarboxylase (EC 4.1.1.4) is only found in those organisms which are capable of producing acetone.

In one preferred embodiment the organism employed in the method according to the invention is an organism, preferably a microorganism, which naturally has the capacity to produce acetone. Thus, preferably the microorganism belongs to the genus Clostridium, Bacillus or Pseudomonas, more preferably to the species Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium cellulolyticum, Bacillus polymyxa or Pseudomonas putida.

In a further preferred embodiment, the organism employed in the method according to the invention is an organism, preferably a microorganism, which naturally has the capacity to produce acetone and which is recombinant in the sense that it has further been genetically modified so as to express an enzyme as defined above. The term “recombinant” in one embodiment means that the organism is genetically modified so as to contain a foreign nucleic acid molecule encoding an enzyme as defined above. In a preferred embodiment the organism has been genetically modified so as to contain a foreign nucleic acid molecule encoding an enzyme as defined above, e.g. a HMG CoA synthase, a C—C bond cleavage/condensation lyase, such as a HMG CoA lyase, or a PksG protein or a foreign nucleic acid sequence encoding any possible combination of such proteins. The term “foreign” in this context means that the nucleic acid molecule does not naturally occur in said organism/microorganism. This means that it does not occur in the same structure or at the same location in the organism/microorganism. In one preferred embodiment, the foreign nucleic acid molecule is a recombinant molecule comprising a promoter and a coding sequence encoding the respective enzyme, e.g. a HMG CoA synthase and/or a C—C bond cleavage/condensation lyase, such as HMG CoA lyase, and/or a PksG protein, in which the promoter driving expression of the coding sequence is heterologous with respect to the coding sequence. Heterologous in this context means that the promoter is not the promoter naturally driving the expression of said coding sequence but is a promoter naturally driving expression of a different coding sequence, i.e., it is derived from another gene, or is a synthetic promoter or a chimeric promoter. Preferably, the promoter is a promoter heterologous to the organism/microorganism, i.e. a promoter which does not naturally occur in the respective organism/microorganism. Even more preferably, the promoter is an inducible promoter. Promoters for driving expression in different types of organisms, in particular in microorganisms, are well known to the person skilled in the art.

In another preferred embodiment the nucleic acid molecule is foreign to the organism/microorganism in that the encoded enzyme(s), e.g. the HMG CoA synthase and/or the encoded C—C bond cleavage/condensation lyase, such as HMG CoA lyase, and/or PksG protein, is/are not endogenous to the organism/microorganism, i.e. are naturally not expressed by the organism/microorganism when it is not genetically modified. In other words, the encoded HMG CoA synthase and/or the encoded C—C bond cleavage/condensation lyase, such as HMG CoA lyase, and/or PksG protein is/are heterologous with respect to the organism/microorganism.

The term “recombinant” in another embodiment means that the organism is genetically modified in the regulatory region controlling the expression of an enzyme as defined above which naturally occurs in the organism so as to lead to an increase in expression of the respective enzyme in comparison to a corresponding non-genetically modified organism. The meaning of the term high “higher expression” is described further below.

Such a modification of a regulatory region can be achieved by methods known to the person skilled in the art. One example is to exchange the naturally occurring promoter by a promoter which allows for a higher expression or to modify the naturally occurring promoter so as to show a higher expression. Thus, in this embodiment the organism contains in the regulatory region of the gene encoding an enzyme as defined above a foreign nucleic acid molecule which naturally does not occur in the organism and which leads to a higher expression of the enzyme in comparison to a corresponding non-genetically modified organism.

The foreign nucleic acid molecule may be present in the organism/microorganism in extrachromosomal form, e.g. as plasmid, or stably integrated in the chromosome. A stable integration is preferred.

In a further preferred embodiment the organism/microorganism is characterized in that the expression/activity of an enzyme as defined above, e.g. of a HMG CoA synthase and/or a C—C bond cleavage/condensation lyase, such as HMG CoA lyase, and/or a PksG protein, is higher in the organism/microorganism genetically modified with the foreign nucleic acid molecule in comparison to the corresponding non-genetically modified organism/microorganism. A “higher” expression/activity means that the expression/activity of the enzyme, in particular of the HMG CoA synthase and/or a C—C bond cleavage/condensation lyase, such as HMG CoA lyase, and/or a PksG protein, in the genetically modified microorganism is at least 10%, preferably at least 20%, more preferably at least 30% or 50%, even more preferably at least 70% or 80% and particularly preferred at least 90% or 100% higher than in the corresponding non-genetically modified organism/microorganism. In even more preferred embodiments the increase in expression/activity may be at least 150%, at least 200% or at least 500%. In particularly preferred embodiments the expression is at least 10-fold, more preferably at least 100-fold and even more preferred at least 1000-fold higher than in the corresponding non-genetically modified organism/microorganism.

The term “higher” expression/activity also covers the situation in which the corresponding non-genetically modified organism/microorganism does not express a corresponding enzyme, e.g. a HMG CoA synthase and/or a C—C bond cleavage/condensation lyase, such as a HMG CoA lyase, and/or a PksG protein, so that the corresponding expression/activity in the non-genetically modified organism/microorganism is zero.

Methods for measuring the level of expression of a given protein in a cell are well known to the person skilled in the art. In one embodiment, the measurement of the level of expression is done by measuring the amount of the corresponding protein. Corresponding methods are well known to the person skilled in the art and include Western Blot, ELISA etc. In another embodiment the measurement of the level of expression is done by measuring the amount of the corresponding RNA. Corresponding methods are well known to the person skilled in the art and include, e.g., Northern Blot.

Methods for measuring the enzymatic activity of the above-mentioned enzymes, in particular HMG CoA synthase and/or a C—C bond cleavage/condensation lyase, such as a HMG CoA lyase, and/or a PksG protein, respectively, are known in the art and have already been described above.

In another preferred embodiment, the organism employed in the method according to the invention is a genetically modified organism, preferably a microorganism, derived from an organism/microorganism which naturally does not produce acetone but which has been genetically modified so as to produce acetone, i.e. by introducing the gene(s) necessary for allowing the production of acetone in the organism/microorganism. In principle any microorganism can be genetically modified in this way. The enzymes responsible for the synthesis of acetone have been described above. Genes encoding corresponding enzymes are known in the art and can be used to genetically modify a given microorganism so as to produce acetone. As described above, the reaction steps 1 and 2 of the acetone synthesis occur naturally in most organisms. However, reaction step 3 is characteristic and crucial for acetone synthesis. Thus, in a preferred embodiment, a genetically modified organism/microorganism derived from an organism/microorganism which naturally does not produce acetone is modified so as to contain a nucleotide sequence encoding an enzyme catalyzing the conversion of acetoacetate into acetone by decarboxylation, e.g. an acetoacetate decarboxylase (EC 4.1.1.4). Nucleotide sequences from several organisms encoding this enzyme are known in the art, e.g. the adc gene from Clostridium acetobutylicum (Uniprot accession numbers P23670 and P23673), Clostridium beijerinckii (Clostridium MP; Q9RPK1), Clostridium pasteurianum (Uniprot accession number P81336), Bradyrhizobium sp. (strain BTAi1/ATCC BAA-1182; Uniprot accession number A5EBU7), Burkholderia mallei (ATCC 10399 A9LBS0), Burkholderia mallei (Uniprot accession number A3MAE3), Burkholderia mallei FMH A5XJB2, Burkholderia cenocepacia (Uniprot accession number A0B471), Burkholderia ambifaria (Uniprot accession number Q0b5P1), Burkholderia phytofirmans (Uniprot accession number B2T319), Burkholderia spec. (Uniprot accession number Q38ZU0), Clostridium botulinum (Uniprot accession number B2TLN8), Ralstonia pickettii (Uniprot accession number B2UIG7), Streptomyces nogalater (Uniprot accession number Q9EYI7), Streptomyces avermitilis (Uniprot accession number Q82NF4), Legionella pneumophila (Uniprot accession number Q5ZXQ9), Lactobacillus salivarius (Uniprot accession number Q1WVG5), Rhodococcus spec. (Uniprot accession number Q0S7W4), Lactobacillus plantarum (Uniprot accession number Q890G0), Rhizobium leguminosarum (Uniprot accession number Q1M911), Lactobacillus casei (Uniprot accession number Q03B66), Francisella tularensis (Uniprot accession number Q0BLC9), Saccharopolyspora erythreae (Uniprot accession number A4FKR9), Korarchaeum cryptofilum (Uniprot accession number B1L3N6), Bacillus amyloliquefaciens (Uniprot accession number A7Z8K8), Cochliobolus heterostrophus (Uniprot accession number Q8NJQ3), Sulfolobus islandicus (Uniprot accession number C3ML22) and Francisella tularensis subsp. holarctica (strain OSU18).

More preferably, the organism, preferably microorganism, is genetically modified so as to be transformed with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 2 of the acetone synthesis, i.e. the conversion of acetoacetyl CoA into acetoacetate.

Even more preferably, the organism, preferably microorganism, is genetically modified so as to be transformed with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 1 of the acetone synthesis, i.e. the condensation of two molecules of acetyl CoA into acetoacetatyl CoA.

In a particularly preferred embodiment the organism/microorganism is genetically modified so as to be transformed with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 1 of the acetone synthesis and with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 2 of the acetone synthesis or with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 1 of the acetone synthesis and with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 3 of the acetone synthesis or with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 2 of the acetone synthesis and with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 3 of the acetone synthesis or with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 1 of the acetone synthesis and with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 2 of the acetone synthesis and with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 3 of the acetone synthesis.

Methods for preparing the above mentioned genetically modified organism, preferably microorganisms, are well known in the art. Thus, generally, the organism/microorganism is transformed with a DNA construct allowing expression of the respective enzyme in the microorganism. Such a construct normally comprises the coding sequence in question linked to regulatory sequences allowing transcription and translation in the respective host cell, e.g. a promoter and/enhancer and/or transcription terminator and/or ribosome binding sites etc. The prior art already describes microorganisms which have been genetically modified so as to be able to produce acetone. In particular genes from, e.g., Clostridium acetobutylicum have been introduced into E. coli thereby allowing the synthesis of acetone in E. coli, a bacterium which naturally does not produce acetone (Bermejo et al., Appl. Environ. Microbiol. 64 (1998); 1079-1085; Hanai et al., Appl. Environ. Microbiol. 73 (2007), 7814-7818). In particular Hanai et al. (loc. cit.) shows that it is sufficient to introduce a nucleic acid sequence encoding an acetoacetate decarboxylase (such as that from Clostridium acetobutylicum) in order to achieve acetone production in E. coli indicating that the endogenous enzymes in E. coli catalyzing the above-mentioned reaction steps 1 and 2 (i.e. the expression products of the E. coli atoB and atoAD genes) are sufficient to provide substrate for the acetone production.

In a particularly preferred embodiment the organism, preferably a microorganism, employed in the method according to the invention is a recombinant organism/microorganism derived from an organism/microorganism which naturally does not produce acetone but which has been genetically modified, as described above, so as to produce acetone and which expresses an enzyme which is capable of catalyzing the formation of a covalent bond between the carbon atom of the oxo (i.e. the C═O) group of acetone and the carbon atom (C²) corresponding to the methyl group of the compound which provides an activated acetyl group as defined above. The term “recombinant” in this context preferably means that the organism is recombinant in the sense that it has further been genetically modified so as to express an enzyme as defined above. The term “recombinant” in one embodiment means that the organism is genetically modified so as to contain a foreign nucleic acid molecule encoding an enzyme as defined above, e.g. a HMG CoA synthase or a C—C bond cleavage/condensation lyase, such as a HMG CoA lyase, or a PksG protein, or a foreign nucleic acid molecule encoding any possible combination of the above defined enzymes.

As regards the definition of the term “foreign nucleic acid molecule” the same applies what has already been set forth above.

The term “recombinant” in another embodiment means that the organism is genetically modified in the regulatory region controlling the expression of an enzyme as defined above which naturally occurs in the organism so as to lead to an increase in expression of the respective enzyme in comparison to a corresponding non-genetically modified organism. The meaning of the term high “higher expression” is described further below.

Such a modification of a regulatory region can be achieved by methods known to the person skilled in the art. One example is to exchange the naturally occurring promoter by a promoter which allows for a higher expression or to modify the naturally occurring promoter so as to show a higher expression. Thus, in this embodiment the organism contains in the regulatory region of the gene encoding an enzyme as defined above a foreign nucleic acid molecule which naturally does not occur in the organism and which leads to a higher expression of the enzyme in comparison to a corresponding non-genetically modified organism.

Preferably such an organism/microorganism is characterized in that the expression/activity of said enzyme, e.g. the HMG CoA synthase and/or a C—C bond cleavage/condensation lyase, such as a HMG CoA lyase, and/or a PksG protein, is higher in the recombinant organism/microorganism in comparison to the corresponding non-genetically modified organism/microorganism. A “higher” expression/activity means that the expression/activity of the enzyme, e.g. the HMG CoA synthase and/or a C—C bond cleavage/condensation lyase, such as HMG CoA lyase, and/or a PksG protein, in the genetically modified organism/microorganism is at least 10%, preferably at least 20%, more preferably at least 30% or 50%, even more preferably at least 70% or 80% and particularly preferred at least 90% or 100% higher than in the corresponding non-genetically modified organism/microorganism. In even more preferred embodiments the increase in expression/activity may be at least 150%, at least 200% or at least 500%. In particularly preferred embodiments the expression is at least 10-fold, more preferably at least 100-fold and even more preferred at least 1000-fold higher than in the corresponding non-genetically modified organism/microorganism.

The term “higher” expression/activity also covers the situation in which the corresponding non-genetically modified organism/microorganism does not express said enzyme, e.g. a HMG CoA synthase and/or a C—C bond cleavage/condensation lyase, such as a HMG CoA lyase, and/or a PksG protein, so that the corresponding expression/activity in the non-genetically modified organism/microorganism is zero.

As regards the methods for measuring the level of expression or activity, the same applies what has already been said above.

The term “organism” as used in the context of the present invention refers in general to any possible type of organism, in particular eukaryotic organisms, prokaryotic organisms and archaebacteria. The term includes animal, plants, fungi, bacteria and archaebacteria. The term also includes isolated cells or cell aggregates of such organisms, like tissue or calli.

In one preferred embodiment, the organism is a microorganism. The term “microorganism” in the context of the present invention refers to prokaryotic cells, in particular bacteria, as well as to fungi, such as yeasts, and also to algae and archaebacteria. In one preferred embodiment, the microorganism is a bacterium. In principle any bacterium can be used. Preferred bacteria to be employed in the process according to the invention are bacteria of the genus Bacillus, Clostridium, Pseudomonas or Escherichia. In a particularly preferred embodiment the bacterium belongs to the genus Escherichia and even more preferred to the species Escherichia coli.

In another preferred embodiment the microorganism is a fungus, more preferably a fungus of the genus Saccharomyces, Schizosaccharomyces, Aspergillus or Trichoderma and even more preferably of the species Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus niger or of the species Trichoderma reesei.

In still another preferred embodiment the microorganism is a photosynthetically active microorganism such as bacteria which are capable of carrying out photosynthesis or micro-algae.

In a particularly preferred embodiment the microorganism is an algae, more preferably an algae belonging to the diatomeae.

If microorganism are used in the context of the method of the present invention, it is also conceivable to carry out the method according to the invention in a manner in which two types of microorganisms are employed, i.e. one type which produces acetone and one type which uses the acetone produced by the first type of microorganisms to convert it with the help of an enzyme as defined herein above.

When the process according to the invention is carried out in vivo by using microorganisms providing the respective enzyme activity/activities, the microorganisms are cultivated under suitable culture conditions allowing the occurrence of the enzymatic reaction(s). The specific culture conditions depend on the specific microorganism employed but are well known to the person skilled in the art. The culture conditions are generally chosen in such a manner that they allow the expression of the genes encoding the enzymes for the respective reactions. Various methods are known to the person skilled in the art in order to improve and fine-tune the expression of certain genes at certain stages of the culture such as induction of gene expression by chemical inducers or by a temperature shift.

In another preferred embodiment the organism employed in the method according to the invention is an organism which is capable of photosynthesis, such as a plant or microalgae. In principle any possible plant can be used, i.e. a monocotyledonous plant or a dicotyledonous plant. It is preferable to use a plant which can be cultivated on an agriculturally meaningful scale and which allows to produce large amounts of biomass. Examples are grasses like Lolium, cereals like rye, barley, oat, millet, maize, other starch storing plants like potato or sugar storing plants like sugar cane or sugar beet. Conceivable is also the use of tobacco or of vegetable plants such as tomato, pepper, cucumber, egg plant etc. Another possibility is the use of oil storing plants such as rape seed, olives etc. Also conceivable is the use of trees, in particular fast growing trees such as eucalyptus, poplar or rubber tree (Hevea brasiliensis).

The present invention also relates to an organism, preferably a microorganism, which is characterized by the following features:

-   -   (a) it is capable of producing acetone; and     -   (b) it expresses an enzyme which is capable of catalyzing the         formation of a covalent bond between the carbon atom of the oxo         (i.e. the C═O) group of acetone and the carbon atom (C²)         corresponding to the methyl group of the compound which provides         an activated acetyl group as defined above, preferably an enzyme         with the activity of a HMG CoA synthase (EC 2.3.3.10) and/or an         enzyme with the activity of a C—C bond cleavage/condensation         lyase, such as a HMG CoA lyase (EC 4.1.3.4) and/or a PksG         protein.

As regards the source, nature, properties, sequence etc. of the enzyme, in particular the HMG CoA synthase, the C—C bond cleavage/condensation lyase, such as HMG CoA lyase, and/or a PksG protein expressed in the organism according to the invention, the same applies as has been set forth above in connection with the method according to the invention.

In one preferred embodiment, the organism according to the invention is an organism, preferably a microorganism which naturally has the capacity to produce acetone, i.e., feature (a) mentioned above is a feature which the organism, preferably microorganism, shows naturally. Thus, preferably the organism is a microorganism which belongs to the genus Clostridium, Bacillus or Pseudomonas, more preferably to the species Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium cellulolyticum, Bacillus polymyxa or Pseudomonas putida.

In another preferred embodiment, the organism, preferably microorganism, according to the invention is a genetically modified organism/microorganism derived from an organism/microorganism which naturally does not produce acetone but which has been genetically modified so as to produce acetone, i.e. by introducing the gene(s) necessary for allowing the production of acetone in the organism/microorganism. In principle any organism/microorganism can be genetically modified in this way. The enzymes responsible for the synthesis of acetone have been described above. Genes encoding corresponding enzymes are known in the art and can be used to genetically modify a given organism, preferably microorganism so as to produce acetone.

In a preferred embodiment, a genetically modified organism/microorganism derived from an organism/microorganism which naturally does not produce acetone is modified so as to contain a nucleotide sequence encoding an enzyme catalyzing the conversion of acetoacetate into acetone by decarboxylation, e.g. an acetoacetate decarboxylase (EC 4.1.1.4). Nucleotide sequences from several organisms encoding this enzyme are known in the art, e.g. the adc gene from Clostridium acetobutylicum. More preferably, the organism/microorganism is genetically modified so as to be transformed with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 2 of the acetone synthesis, i.e. the conversion of acetoacetyl CoA into acetoacetate.

Even more preferably, the organism/microorganism is genetically modified so as to be transformed with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 1 of the acetone synthesis, i.e. the condensation of two molecules of acetyl CoA into acetoacetatyl CoA.

In a particularly preferred embodiment the organism/microorganism is genetically modified so as to be transformed with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 1 of the acetone synthesis and with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 2 of the acetone synthesis or with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 1 of the acetone synthesis and with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 3 of the acetone synthesis or with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 2 of the acetone synthesis and with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 3 of the acetone synthesis or with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 1 of the acetone synthesis and with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 2 of the acetone synthesis and with a nucleic acid molecule encoding an enzyme capable of catalyzing the above mentioned reaction step 3 of the acetone synthesis.

Methods for preparing the above mentioned genetically modified organisms/microorganisms are well known in the art. Thus, generally, the organism/microorganism is transformed with a DNA construct allowing expression of the respective enzyme in the organism/microorganism. Such a construct normally comprises the coding sequence in question linked to regulatory sequences allowing transcription and translation in the respective host cell, e.g. a promoter and/enhancer and/or transcription terminator and/or ribosome binding sites etc. The prior art already describes organism, in particular microorganisms which have been genetically modified so as to be able to produce acetone. In particular genes from, e.g., Clostridium acetobutylicum have been introduced into E. coli thereby allowing the synthesis of acetone in E. coli, a bacterium which naturally does not produce acetone (Bermejo et al., Appl. Environ. Microbiol. 64 (1998); 1079-1085; Hanai et al., Appl. Environ. Microbiol. 73 (2007), 7814-7818). In particular Hanai et al. (loc. cit.) shows that it is sufficient to introduce a nucleic acid sequence encoding an acetoacetate decarboxylase (such as that from Clostridium acetobutylicum) in order to achieve acetone production in E. coli indicating that the endogenous enzymes in E. coli catalyzing the above-mentioned reaction steps 1 and 2 (i.e. the expression products of the E. coli atoB and atoAD genes) are sufficient to provide substrate for the acetone production.

In a further preferred embodiment the organism, preferably a microorganism, according to the invention is genetically modified so as to express an enzyme which is capable of catalyzing the formation of a covalent bond between the carbon atom of the oxo (i.e. the C═O) group of acetone and the carbon atom (C²) corresponding to the methyl group of the compound which provides an activated acetyl group. In this context, the term “recombinant” means in a first aspect that the organism contains a foreign nucleic acid molecule encoding an enzyme which is capable of catalyzing the formation of a covalent bond between the carbon atom of the oxo (i.e. the C═O) group of acetone and the carbon atom (C²) corresponding to the methyl group of the compound which provides an activated acetyl group, preferably a foreign nucleic acid molecule encoding a HMG CoA synthase or a foreign nucleic acid molecule encoding a C—C bond cleavage/condensation lyase, such as a HMG CoA lyase, or a foreign nucleic acid molecule encoding a PksG protein or a foreign nucleic acid molecule encoding any possible combination of the enzymes having the above-mentioned property. The term “foreign” in this context means that the nucleic acid molecule does not naturally occur in said organism/microorganism. This means that it does not occur in the same structure or at the same location in the organism/microorganism. In one preferred embodiment, the foreign nucleic acid molecule is a recombinant molecule comprising a promoter and a coding sequence encoding said enzyme, e.g. the HMG CoA synthase and/or a C—C bond cleavage/condensation lyase, such as a HMG CoA lyase, and/or a PksG protein, in which the promoter driving expression of the coding sequence is heterologous with respect to the coding sequence. Heterologous in this context means that the promoter is not the promoter naturally driving the expression of said coding sequence but is a promoter naturally driving expression of a different coding sequence, i.e., it is derived from another gene, or is a synthetic promoter or a chimeric promoter. Preferably, the promoter is a promoter heterologous to the organism/microorganism, i.e. a promoter which does naturally not occur in the respective organism/microorganism. Even more preferably, the promoter is an inducible promoter. Promoters for driving expression in different types of organisms, in particular microorganisms, are well known to the person skilled in the art.

In another preferred embodiment the nucleic acid molecule is foreign to the organism/microorganism in that the encoded enzyme(s), e.g. the HMG CoA synthase and/or the encoded C—C bond cleavage/condensation lyase, such as a HMG CoA lyase, and/or the encoded PksG protein, is/are not endogenous to the organism/microorganism, i.e. are naturally not expressed by the organism/microorganism when it is not genetically modified. In other words, the encoded enzyme(s), e.g. the HMG CoA synthase and/or the encoded C—C bond cleavage/condensation lyase, such as HMG CoA lyase, and/or the encoded PksG protein, is/are heterologous with respect to the organism/microorganism.

The term “recombinant” in another aspect means that the organism is genetically modified in the regulatory region controlling the expression of an enzyme as defined above which naturally occurs in the organism so as to lead to an increase in expression of the respective enzyme in comparison to a corresponding non-genetically modified organism. The meaning of the term high “higher expression” is described further below.

Such a modification of a regulatory region can be achieved by methods known to the person skilled in the art. One example is to exchange the naturally occurring promoter by a promoter which allows for a higher expression or to modify the naturally occurring promoter so as to show a higher expression. Thus, in this embodiment the organism contains in the regulatory region of the gene encoding an enzyme as defined above a foreign nucleic acid molecule which naturally does not occur in the organism and which leads to a higher expression of the enzyme in comparison to a corresponding non-genetically modified organism.

In a further preferred embodiment the organism/microorganism is characterized in that the expression/activity of said enzyme, e.g. the HMG CoA synthase and/or a C—C bond cleavage/condensation lyase, such as HMG CoA lyase, and/or the PksG protein, is higher in the organism/microorganism genetically modified with the foreign nucleic acid molecule in comparison to the corresponding non-genetically modified organism/microorganism. A “higher” expression/activity means that the expression/activity of the enzyme, e.g. the HMG CoA synthase and/or a C—C bond cleavage/condensation lyase, such as HMG CoA lyase, and/or the PksG protein, in the genetically modified organism/microorganism is at least 10%, preferably at least 20%, more preferably at least 30% or 50%, even more preferably at least 70% or 80% and particularly preferred at least 90% or 100% higher than in the corresponding non-genetically modified organism/microorganism. In even more preferred embodiments the increase in expression/activity may be at least 150%, at least 200% or at least 500%. In particularly preferred embodiments the expression is at least 10-fold, more preferably at least 100-fold and even more preferred at least 1000-fold higher than in the corresponding non-genetically modified organism/microorganism.

The term “higher” expression/activity also covers the situation in which the corresponding non-genetically modified organism/microorganism does not express a corresponding enzyme, e.g. a HMG CoA synthase and/or a C—C bond cleavage/condensation lyase, such as a HMG CoA lyase, and/or a PksG protein, so that the corresponding expression/activity in the non-genetically modified organism/microorganism is zero.

Methods for measuring the level of expression of a given protein in a cell are well known to the person skilled in the art. In one embodiment, the measurement of the level of expression is done by measuring the amount of the corresponding protein. Corresponding methods are well known to the person skilled in the art and include Western Blot, ELISA etc. In another embodiment the measurement of the level of expression is done by measuring the amount of the corresponding RNA. Corresponding methods are well known to the person skilled in the art and include, e.g., Northern Blot.

Methods for measuring the enzymatic activity of the above-mentioned enzyme, in particular of a HMG CoA synthase and/or a HMG CoA lyase and/or a PksG protein, respectively, are known in the art and have already been described above.

The term “organism” as used in the context of the present invention refers in general to any possible type of organism, in particular eukaryotic organisms, prokaryotic organisms and archaebacteria. The term includes animal, plants, fungi, bacteria and archaebacteria. The term also includes isolated cells or cell aggregates of such organisms, like tissue or calli.

In one preferred embodiment, the organism is a microorganism. The term “microorganism” in the context of the present invention refers to prokaryotic cells, in particular bacteria, as well as to fungi, such as yeasts, and also to algae and archaebacteria. In one preferred embodiment, the microorganism is a bacterium. In principle any bacterium can be used. Preferred bacteria to be employed in the process according to the invention are bacteria of the genus Bacillus, Clostridium, Pseudomonas or Escherichia. In a particularly preferred embodiment the bacterium belongs to the genus Escherichia and even more preferred to the species Escherichia coli.

In another preferred embodiment the microorganism is a fungus, more preferably a fungus of the genus Saccharomyces, Schizosaccharomyces, Aspergillus or Trichoderma and even more preferably of the species Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus niger or of the species Trichoderma reesei.

In still another preferred embodiment the microorganism is a photosynthetically active microorganism such as bacteria which are capable of carrying out photosynthesis or micro-algae.

In a particularly preferred embodiment the microorganism is an algae, more preferably an algae from the genus belonging to the diatomeae.

In another preferred embodiment the organism according to the invention is an organism which is capable of photosynthesis, such as a plant or micro-algae. In principle, it can be any possible plant, i.e. a monocotyledonous plant or a dicotyledonous plant. It is preferably a plant which can be cultivated on an agriculturally meaningful scale and which allows to produce large amounts of biomass. Examples are grasses like Lolium, cereals like rye, barley, oat, millet, maize, other starch storing plants like potato or sugar storing plants like sugar cane or sugar beet. Conceivable is also the use of tobacco or of vegetable plants such as tomato, pepper, cucumber, egg plant etc. In another preferred embodiment the plant is an oil storing plants such as rape seed, olives etc. Also conceivable is the use of trees, in particular fast growing trees such as eucalyptus, poplar or rubber tree (Hevea brasiliensis).

The present invention also relates to the use of an organism, preferably a microorganism, which is characterized by the following features:

-   -   (a) it is capable of producing acetone; and     -   (b) it expresses an enzyme which is capable of catalyzing the         formation of a covalent bond between the carbon atom of the oxo         (i.e. the C═O) group of acetone and the carbon atom (C²)         corresponding to the methyl group of the compound which provides         an activated acetyl group as defined herein above, preferably an         enzyme with the activity of a HMG CoA synthase (EC 2.3.3.10)         and/or an enzyme with the activity of a C—C bond         cleavage/condensation lyase, such as a HMG CoA lyase (EC         4.1.3.4), and/or a PksG protein for the production of         3-hydroxy-3-methylbutyric acid.

I.e., the present invention also relates to the use of an organism/microorganism according to the invention for the production of 3-hydroxy-3-methylbutyric acid.

The present invention also relates to a composition comprising an organism according to the present invention.

Moreover, the present invention also relates to a composition comprising (i) acetone; and (ii) a compound which provides an activated acetyl group as defined herein above; and (iii) an enzyme which is capable of catalyzing the formation of a covalent bond between the carbon atom of the oxo (i.e. the C═O) group of acetone and the carbon atom (C²) corresponding to the methyl group of the compound which provides an activated acetyl group as defined herein above.

For the preferred embodiments of the enzyme the same applies as has already been set forth above in connection with the method and the organism according to the invention.

Moreover, the present invention also relates to the use of an enzyme which is capable of catalyzing the formation of a covalent bond between the carbon atom of the oxo (i.e. the C═O) group of acetone and the carbon atom (C²) corresponding to the methyl group of the compound which provides an activated acetyl group as defined herein above for the production of 3-hydroxy-3-methylbutyric acid.

For the preferred embodiments of the enzyme the same applies as has already been set forth above in connection with the method and the organism according to the invention.

Finally, the present invention also relates to the use of acetone for the production of 3-hydroxy-3-methylbutyric acid, comprising the enzymatic conversion of acetone and a compound which provides an activated acetyl group as defined herein above. In a preferred embodiment the enzymatic conversion is achieved by an enzyme as described above in connection with the method according to the invention, more preferably with an enzyme having the enzymatic activity of a HMG CoA synthase and/or with an enzyme having the enzymatic activity of a C—C bond cleavage/condensation lyase, such as a HMG CoA lyase, and/or a PksG protein, and most preferably the conversion is achieved by the use of an organism according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Chemical structure of 3-hydroxy-3-methylbutyric acid (also referred to as beta-hydroxyisovalerate)

FIG. 2: Reaction scheme of the reaction catalysed by HMG-CoA synthase

FIG. 3: Reaction scheme of the reaction catalysed by HMG-CoA lyase

FIG. 4: Reaction schemes of the reactions of the pksX pathway including the reaction catalysed by the PksG protein

FIG. 5: Reaction scheme of the reaction of the conversion of acetone and a compound containing an activated acetyl group into 3-hydroxy-3-methylbutyric acid

-   -   X stands for         S—CH₂—CH₂—NH—CO—CH₂—CH₂—NH—CO—CH(OH)—C(CH₃)₂—CH₂—O—PO₂H—O—PO₂H—C₁₀H₁₃N₅O₇P         (coenzyme A),         S—CH₂—CH₂—NH—CO—CH₂—CH₂—NH—CO—CH(OH)—C(CH₃)₂—CH₂—O—PO₂H-polypeptide         (acyl-carrier protein),         S—CH₂—CH₂—NH—CO—CH₂—CH₂—NH—CO—CH(OH)—C(CH₃)₂—CH₂—OH         (pantetheine), S—CH₂—CH₂—NH—CO—CH₃ (N-acetyl-cysteamine), S—CH₃         (methane thiol), S—CH₂—CH(NH₂)—CO₂H (cysteine),         S—CH₂—CH₂—CH(NH₂)—CO₂H (homocysteine),         S—CH₂—CH(NH—C₅H₈NO₃)—CO—NH—CH₂—CO₂H (glutathione),         S—CH₂—CH₂—SO₃H (coenzyme M) and OH (acetic acid).

FIG. 6: Mass spectra of commercial available 3-hydroxy-3-methylbutyrate

FIG. 7: Mass spectra of formation of 3-hydroxy-3-methylbutyrate from acetyl-CoA and acetone in the presence of Hmg-CoA synthase from Gallus gallus (P23228).

FIG. 8: Mass spectra of the control assay without enzyme.

FIG. 9: Michaelis-Menten plot for the reaction with the HMG CoA synthase of S. epidermidis described in Example 7

EXAMPLES

The following examples serve to illustrate the invention.

Example 1 Bioinformatic Method Used to Create HMG-CoA Synthases and HMG-CoA Lyases Database

A panel of 12 HMG-CoA synthases and 8 HMG-CoA lyases were selected to create a non-redundant set of proteins aiming to represent the diversity of these enzyme classes as found across eukaryotic organisms. These proteins were identified by performing multiple sequence-based and text-based searches on the Universal Protein Resource Database Uniprot (www.uniprot.org/). They all contain unique features such as conserved protein domains and motifs characteristic to the enzyme class of interest. In order to effectively cover the sequence diversity without having to screen a large set of proteins, the initial pool of enzymes was narrowed down by grouping them into clusters of sequences with more than 85% homology and then selecting one single candidate sequence representative of each cluster. Protein sequence identity ranged from 30% to 80% and from 50% to 80% between any two proteins from the HMG-CoA synthases panel and the lyases panel respectively.

The same approach was applied to select the HMG-CoA synthases and HMG-CoA lyases from prokaryotic organisms. The created set contained 50 proteins homologues to HMG-CoA synthases, including pksG proteins, and 59 proteins homologues to HMG-CoA lyases.

Example 2 Cloning, Expression and Purification of a Collection of HMG-CoA Lyases and HMG-CoA Synthases

Gene Cloning:

The nucleic acid sequences coding for HMG-CoA synthase and lyase from eukaryotic organism were optimized for E. coli codon preference and the genes were obtained by chemical synthesis (GeneArt®, reagents).

The genes encoding for HMG-CoA synthases and lyases from prokaryotic organisms were cloned from genomic DNA of different origins by routine recombinant techniques. These genes were then inserted in a His-tag containing pET 25b and pET 22b vectors (Novagen, Inc.), respectively, for eukaryotic and prokaryotic organisms.

Overexpression in E. Coli:

Plasmids are electroporated into E. coli BL21 bacteria (Novagen) that are then spread on an ampicillin containing LB-Agar Petri dish. The cultures are grown at 30° C. on TB medium, containing 0.5 M sorbitol, 5 mM betaine, 100 μg/ml ampicillin under moderate shaking. When OD (600 nm) reached 0.8, IPTG is added to a final concentration of 1 mM, and expression is run for 16 hours at 20° C. under moderated shaking. The bacteria cells are then harvested by centrifugation at 4° C., 10.000 rpm, 20 minutes and frozen at −80° C.

Cell Extract Preparation:

Cell extracts are prepared by resuspending 1.6 g of cell pellet in 5 ml 50 mM Na₂HPO₄ buffer, containing 300 mM NaCl, 5 mM MgCl₂, 1 mM DTT pH 8. 20 μl lysonase (Novagen) is then added to the preparations, which are incubated for 10 min at room temperature and 20 min on ice. The cell lysis is achieved by triple sonication treatment of 5 minutes in ultrasonic water-bath on ice and homogenization of extract between each pulse. The crude extracts are then clarified by centrifugation at 4° C., 10.000 rpm, 20 minutes.

Protein Purification:

The clear supernatants are loaded onto the PROTINO-1000® Ni-IDA column (columns for the purification of proteins, Macherey-Nagel) which enables the specific immobilisation of proteins carrying 6-histidine tails. The columns are washed and the enzymes are eluted with 4 ml 50 mM Na₂HPO₄ buffer, containing 300 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 250 mM imidazole pH 8. The enzyme containing fractions are then concentrated and desalted on Amicon® Ultra-4 10 kDa filter unit (membranes for filtration, dialysis; Millipore) and resuspended in 250 μl 40 mM Tris-HCl pH8, containing 0.5 mM DTT. The protein concentration is determined by the Bradford method.

The homogeneity of purified enzymes varied from 20% to 75%.

Example 3 Measure of the HMG-CoA Synthase Activity Using Natural Substrates Acetoacetyl-CoA and Acetyl-CoA

The HMG-CoA synthase activity is measured according to Clinkenbeard et al. (J. Biol. Chem. 250 (1975), 3108-3116). The standard assay medium mixture for HMG-CoA synthases contains 40 mM Tris-HCl pH 8, 1 mM MgCl₂, 100 μM acetoacetyl-CoA, 200 μM acetyl-CoA, 0.5 mM DTT in a total volume of 1 ml. Mitochondria HMG-CoA synthases are assayed in the absence of MgCl₂ to avoid the inhibition observed for this enzyme (Reed et al., J. Biol. Chem. 250 (1975), 3117-3123). Reaction is initiated by addition of 0.02 mg/mL enzyme.

A Control assay was carried out in the absence of enzyme. HMG-CoA synthase activity was measured by monitoring the decrease in absorbance at 303 nm that accompanies the acetyl-CoA-dependent disappearance of the enolate form of acetoacetyl-CoA. To account for non-specific disappearance of acetoacetyl-CoA, results obtained in a control assay lacking enzyme were subtracted from results obtained in test samples. The apparent absorption coefficient for acetoacetyl-CoA under the assay conditions was 5600 M⁻¹·cm⁻¹. One enzyme unit represented the disappearance in 1 min of 1 μmol of acetoacetyl-CoA.

TABLE 1 Physiological activity of some purified HMG-CoA synthases or enzymes homologous to HMG CoA synthases Physiological Uniprot activity Accession μmol/min · mg number Organism protein P54961 Blattella germanica (German cockroach) 0.02 P23228 Gallus gallus (Chicken) 0.02 Q01581 Homo sapiens (Human) 0.03 P54873 Arabidopsis thaliana 1.19 P54871 Caenorhabditis elegans 0.23 P54874 Schizosaccharomyces pombe (Fission yeast) 0.61 P54839 Saccharomyces cerevisiae (Baker's yeast) 0.28 P54872 Dictyostelium discoideum (Slime mold) 0.09 Q86HL5 Dictyostelium discoideum (Slime mold) 0.02 Q9M6U3 Brassica juncea 0.02 A5FM54 Flavobacterium johnsoniae 0.02 Q03WZ0 Leuconostoc mesenteroides 0.28 Q2NHU7 Methanosphaera stadtmanae 0.02 Q8CN06 Staphylococcus epidermidis 0.07 Q03QR0 Lactobacillus brevis 0.18 A6UPL1 Methanosarcina mazei 0.01 B2HGT6 Mycobacterium marinum 0.01 Q4L958 Staphylococcus haemolyticus 0.18 Q4A0D6 Staphylococcus saprophyticus 0.08 Q1GAH5 Lactobacillus delbrueckii 0.32

Example 4 Measuring of the HMG-CoA Lyase Activity Using Natural Substrate HMG-CoA

HMG-CoA lyase activity is measured according to Mellanby J et al. (Methods of Enzymatic Analysis; Bergmeyer Ed. (1963), 454-458). The complete reaction mixture (1 ml) containing 40 mM Tris-HCl pH 8, 1 mM MgCl₂, 0.5 mM DTT, 0.4 mM HMG-CoA, 0.2 mM NADH, 5 units of 3-hydroxybutyrate dehydrogenase is incubated for 5 min before adding 0.005 mg/ml of HMG-CoA lyase and then the progress of the reaction is monitored by the decrease in absorbance at 340 nm. A control assay was carried out in the absence of enzyme.

To account for non-specific disappearance of NADH, results obtained in a control assay lacking enzyme were subtracted from results obtained in test samples. Specific activities were calculated as Δμmol NADH/min·mg protein.

TABLE 2 Physiological activity of some purified HMG-CoA lyases Physiological Uniprot activity Accession μmol/min · mg number Organism protein A8WG57 Danio rerio (Zebrafish) (Brachydanio rerio) 4.05 Q29448 Bos taurus (Bovine) 5.79 B6U7B9 Zea mays 13.31 A5FHS2 Flavobacterium johnsoniae 2.89 A1VJH1 Polaromonas naphthalenivorans 34.92 A9IFQ7 Bordetella petrii 9.84 A9IR28 Bordetella petrii 1.74 A1VT25 Polaromonas naphthalenivorans 0.39

Example 5 3-hydroxy-3-methylbutyrate Production

The complete reaction for 3-hydroxy-3-methylbutyrate synthesis contained 40 mM Tris-HCl pH 8, 5 to 50 mM acetyl-CoA, 100 to 500 mM acetone, 1 MgCl₂ (except for mitochondria HMG-CoA synthase), 0.5 mM DTT and enzyme varying in the range from 0.2 to 8 mg/ml. Control reactions were carried in the absence of enzyme and one of the substrates.

The progress of synthesis was followed by analyzing aliquots taken after increasing period of incubation at 30 or 37° C. Typically, an aliquot of 50 μl was removed after 48 h of incubation, heated for 1 min at 100° C. to eliminate the proteins, centrifuged and the supernatant was transferred to a clean vial for HIV detection by mass spectrometry. A solution of 3-hydroxy-3-methylbutyrate was prepared in 40 mM Tris-HCl pH 8, 1 mM MgCl₂, 0.5 mM DTT, heated as described early and used as reference.

The samples were analyzed on a PE SCIEX® API 2000 triple quadrupole mass spectrometer (mass spectrometer, Perkin-Elmer) in negative ion mode with H₂0/acetonitrile=60/40 containing 0.1% triethylamine as mobile phase, flow rate was 40 μl/min. 10 μl of each supernatant were mixed with an equal quantity of mobile phase and directly injected into the mass spectrometer. The presence of [3-hydroxy-3-methylbutyrate-H]⁻ ion was monitored.

A peak corresponding to 3-hydroxy-3-methylbutyrate was observed for the following enzymes:

-   Blattella germanica (German cockroach) P54961 (SEQ ID NO: 6) -   Gallus gallus (Chicken) P23228 (SEQ ID NO: 7) -   Homo sapiens (Human) Q01581 (SEQ ID NO: 8) -   Arabidopsis thaliana P54873 (CAA58763) (SEQ ID NO: 4) -   Caenorhabditis elegans P54871 (SEQ ID NO: 1) -   Schizosaccharomyces pombe (Fission yeast) P54874 (SEQ ID NO: 2) -   Saccharomyces cerevisiae (Baker's yeast) P54839 (SEQ ID NO: 3) -   Dictyostelium discoideum (Slime mold) Q86HL5 (SEQ ID NO: 10) -   Leuconostoc mesenteroides Q03WZ0 (SEQ ID NO:) -   Staphylococcus epidermidis Q8CN06 (SEQ ID NO: 11) -   Lactobacillus delbrueckii Q1GAH5 (SEQ ID NO: 24) -   Staphylococcus haemolyticus Q4L958 (I98>V difference compared to     wild type protein) (SEQ ID NO: 25)

FIGS. 6 to 8 show representative results for commercially available 3-hydroxy-3-methylbutyrate, for the reaction using the HMG CoA synthase from Gallus gallus (P23228) and for the control assay without enzyme.

Example 6 3-hydroxy-3-methylbutyryl-CoA Production Using Lyases

3-hydroxy-3-methylbutyryl-CoA synthesis is carried out in the presence of radiolabeled [2-¹⁴C] acetone. The complete reaction for 3-hydroxy-3-methylbutyryl-CoA synthesis contains 40 mM Tris-HCl pH 8, 5 to 50 mM acetyl-CoA, 100 to 500 mM acetone, 1 to 10 mM MgCl₂, 0.5 mM DTT and enzyme varying in the range from 0.5 to 7 mg/ml. The formation of product is analyzed after separation of reaction mixture by TLC or HPLC.

3-hydroxy-3-methylbutyryl-CoA is also analyzed by TLC method (Stadtman E. R., J. Biol. Chem. 196 (1952), 535-546). An aliquot of reaction is deposited on a cellulose plate and chromatographied in the following solvent system: ethanol/0.1 M sodium acetate pH 4.5 (1/1). Co-A and acetyl-CoA are used as internal standards. R_(f) reported for 3-hydroxy-3-methylbutyryl-CoA is 0.88.

Example 7 Kinetic Parameters for the Enzymatic Reaction Between Acetyl-CoA and Acetone in the Case of HMG Synthases

The kinetic parameters were measured using a variable concentration of acetone and a constant concentration of acetyl-CoA (10 mM) in following conditions:

-   40 mM Tris-HCl pH 8 -   2 mM MgCl₂ -   0-1 M acetone

The final pH was adjusted to 8.

The reaction was initiated by the addition of 3 mg of purified enzyme to the 1 ml reaction mixture. The mixture was then incubated without shaking at 37° C. for 40 h.

Analysis of 3-hydroxy-3-methylbutyrate Production

Thermochemical conditions leading to the decomposition of 3-hydroxy-3-methylbutyrate into isobutene were applied (Pressman et al., JACS, 1940, 2069-2080): the pH of the reaction mixtures was first adjusted to pH 4 using 6N HCl and the samples were then transferred into gas chromatography vials (Interchim). The vials were sealed and incubated at 110° C. for 4 hours, thus leading to the decomposition of 3-hydroxy-3-methylbutyrate into isobutene.

The calibration curve was prepared in the same conditions using commercial 3-hydroxy-3-methylbutyrate.

One milliliter of headspace gas was collected and injected into a HP5890 gas chromatograph (HP) equipped with a FID detector and a CP SilicaPlot® column (chromatography column; Varian). Commercial isobutene was used as reference. From the isobutene signal the amount of 3-hydroxy-3-methylbutyrate initially present in the sample was calculated.

The kinetics parameters for several of the studied HMG-CoA synthases are presented in the following Table.

K_(M) for k_(cat) × 10⁻⁴, k_(cat)/K_(M) × 10⁻⁶, Organism acetone, mM sec⁻¹ mM⁻¹ × sec−¹ Gallus gallus 250 5 2 Staphylococcus 200 0.6 0.3 epidermidis Schizosaccharomyces 200 0.2 0.1 pombe

For the enzyme from S. epidermidis FIG. 9 shows a corresponding Michaelis-Menten plot. 

The invention claimed is:
 1. A method for the production of 3-hydroxy-3-methylbutyric acid consisting essentially of: (a) providing a recombinant microorganism which has been transformed with a polynucleotide encoding 3-hydroxy-3-methylglutaryl-CoA (HMG CoA) synthase and a polynucleotide encoding acetoacetate decarboxylase, wherein at least one of the polynucleotides is heterologous to the recombinant microorganism; and (b) incubating, in the recombinant microorganism (i) acetone and (ii) a compound of formula (I) which provides an activated acetyl group:

wherein X is S—CH₂—CH₂—NH—CO—CH₂—CH₂—NH—CO—CH(OH)—C(CH₃)₂—CH₂—O—PO₂H—PO₂H—C₁₀H₁₃N₅O₇P (coenzyme A), with (c) the HMG CoA synthase, under conditions in which the recombinant microorganism produces acetone and expresses the HMG CoA synthase, thereby producing 3-hydroxy-3-methylbutyric acid.
 2. The method of claim 1, wherein the microorganism naturally has the capacity to produce acetone.
 3. The method of claim 2, wherein the microorganism is of the genus Clostridium, Bacillus or Pseudomonas.
 4. The method of claim 3, wherein the microorganism is selected from the species Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium cellulolyticum, Bacillus polymyxa, and Pseudomonas putida.
 5. The method of claim 1, wherein the microorganism is of a species which naturally does not produce acetone.
 6. The method of claim 5, wherein the microorganism is capable of photosynthesis.
 7. The method of claim 1, wherein the microorganism is Escherichia coli.
 8. The method of claim 1, wherein the microorganism is selected from Saccharomyces, Schizosaccharomyces, Aspergillus and Trichoderma. 